Serum Regulation of Inhibitor of DNA Binding/Differentiation 1 Expression by a BMP Pathway and BMP Responsive El

Immediate Early Genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP Responsive Element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs

DNA bending in the ternary nucleoprotein complex at the c-fos promoter.

Transcriptional induction of the c-fos proto-oncogene in response to serum growth factors is mediated in part by a ternary complex that forms on the serum response element (SRE) within its promoter. This complex consists of Elk-1, serum response factor (SRF) and the SRE. Elk-1 is phosphorylated by MAP kinase, which correlates with the induction of c-fos transcription. In this study we have investigated the protein-induced DNA bending which occurs during the formation and post-translational modification of the ternary complex that forms at the c-fos SRE. Circular permutation analysis demonstrates that the minimal DNA-binding domain of SRF, which contains the MADS box, is sufficient to induce flexibility into the centre of its binding site within the SRE. Phasing analysis indicates that at least part of this flexibility results in the production of a directional bend towards the minor groove. The isolated ETS domains from Elk-1 and SAP-1 induce neither DNA bending nor increased DNA flexibility. Formation of ternary complexes by binding of Elk-1 to the binary SRF:SRE complex results in a change in the flexibility of the SRE. Phosphorylation of Elk-1 by MAP kinase (p42/ERK2) induces further minor changes in this DNA flexibility. However, phasing analysis reveals that the recruitment of Elk-1 to form the ternary complex affects the SRF-induced directional DNA bend in the SRE. The potential roles of DNA bending at the c-fos SRE are discussed

Molecular study of SRF-cofactor interactions.

Serum Response Factor regulates a large array of genes involved in diverse processes including cell proliferation, muscle differentiation and development, and cytoskeletal processes such as cell migration and adhesion. The specificity and versatility of the SRF responses is achieved by combinatorial interactions with accessory factors. SRF binds to the CC(A/T)2A(A/T)3GG CArG box consensus sequence within the promoters of its target genes and acts as a docking platform for diverse signal regulated and cell- type specific cofactors to elicit their distinct responses. In fibroblasts two pathways signal through SRF in a mutually exclusive manner. MAP kinase signalling results in transcriptional activation of a subset of SRF target genes, via the interaction of SRF with members of the TCF family of Ets domain proteins. In contrast Rho-signalling induced changes in actin dynamics result in the association of SRF with members of the Myocardin-related family of SRF cofactors (MAL/MRTF-A/MKL1 and MAL16/MRTF-B/MKL2). The results described in this thesis characterise the molecular mechanism of MAL-SRF complex formation. MAL binds SRF as a dimer via a seven-residue core sequence within the MAL B1 region. Residues in the neighbouring Q-box enhance MAL-SRF complex formation, although these do not contact SRF directly. The MAL-SRF interaction displays the properties of a Rho-regulated cofactor. MAL competes with TCF for SRF binding due to the interaction of both cofactors with the same hydrophobic groove and pocket on SRF. In contrast to TCF, MAL-SRF complex formation depends on the intact N-terminus of the SRF DNA-binding domain. Mutations in the SRF al-helix that reduce DNA bending also impair complex formation with MAL. These mutations however do not affect DNA distortion in the MAL-SRF complex. Efficient MAL-SRF complex formation requires that SRF be bound to its cognate DNA and that MAL directly contacts DNA on either side of the CArG box. My results support a model in which each MAL monomer adds a p-strand consisting of the core B1 sequence, to the p-sheet of the SRF DNA-binding domain in a similar way to TCF, while also making direct DNA contacts in the ternary complex facilitated by SRF- induced DNA distortion. My analysis of complex formation between MAL and SRF demonstrates that members of the MRTF and TCF families of SRF cofactors interact with SRF using related but distinct mechanisms, thus providing a molecular rationale for their mutually exclusive transcriptional responses and the specificity of signalling to SRF

Structural and functional analysis of the IkBalpha protein

NF-KB/Rel transcription factors and IKB proteins play a central role in the rapid induction of genes transcribed in response to a variety of extracellular stimuli. Signal induction results in the phosphorylation and ubiquitination of IKBalpha a prior to proteasomal degradation and release of NF-KB. The objective of this work was to identify residues within the N- and C-termini of IKBalpha which were important for the function of the protein, specifically the residues contacting the p65 subunit of NF- KB and those involved in the turnover of IKBalpha. In addition, the structure of the C-terminal region of IKBalpha was also examined to gain a better understanding of the functional properties of this domain. The identification of conserved stretches of acidic residues in the C-terminal region of IKBalpha prompted the suggestion that perhaps these amino acids were important for interacting with the positively charged nuclear localisation signal of the p65 subunit of NF- KB. Accordingly two regions, residues 284-286 and residues 300-302 (glutamic acid, aspartic acid, glutamic acid in both regions), were targeted for mutation (and referred to as the C-terminal mutants) to examine their role, if any, in IKBalpha -p65 association. A second set of mutants were generated following a protease sensitivity study on IKBalpha which revealed that residues 251 (tyrosine), 258 (tryptophan) and 275 (glutamic acid) were protected from digestion in the presence of p65. These amino acids were located in the low homology sixth ankyrin repeat of IKBalpha, thought to act as a flexible linker region between the highly conserved central five ankyrin repeats and the C-terminal region of the protein. Consequently, amino acids 258 and 275 were selected for mutation (referred to as the linker mutants). The in vitro characterisation of the C-terminal and linker mutants demonstrated that neither amino acids 258 and 275 nor amino acids 284-286 and 300-302 affected the ability of IKBalpha to interact with p65 homodimers, even under conditions of varying pH or ionic strength. However, residues 284-286 reduced the inhibitory capacity of IKBalpha with respect to p65 homodimers. The results indicated that the region of IKBalpha required for the inhibition of p65 DNA binding activity, possibly located in or around residues 284-286, was separable from the area of the protein responsible for association with p65. Attempts to phosphorylate the C-terminal and linker mutants in vitro using either casein kinase I or casein kinase II demonstrated that the C-terminal mutants were not efficiently phosphorylated by casein kinase II but were phosphorylated by casein kinase I. Both kinases were shown to phosphorylate wild-type IKBalpha and the linker mutants. Therefore, residues 284-286 and 300-302 were possibly important for the in vivo phosphorylation of IKBalpha through casein kinase II. The expression of the C-terminal and linker mutants from vectors transiently transfected into 293 cells revealed that residues 284-286 and residues 300-302 were required for either inducible or constitutive degradation of IKBalpha, but more likely constitutive turnover. All mutants appeared to undergo signal-induced ubiquitination. Furthermore, the mutants were capable of interacting with p65 and in a different cell line (Cos7 cells) all appeared to allow NF-KB-dependent transcription from a luciferase reporter. The final result was thought to be a consequence of different degradation characteristics existing within Cos7 cells compared to 293 cells. In the light of recent data, it was concluded that the C-terminal residues of IKBalpha were important for constitutive, rather than inducible turnover of IKBalpha